Apoptosis, or programmed cell death, is a principal mechanism by which organisms eliminate unwanted cells. The deregulation of apoptosis, either excessive apoptosis or the failure to undergo it, has been implicated in a number of diseases such as cancer, acute inflammatory and autoimmune disorders, ischemic diseases and certain neurodegenerative disorders (see Science, 1998, 281, 1283-1312; Ellis et al., Ann. Rev. Cell. Biol., 1991, 7, 663). Caspases are a family of cysteine protease enzymes that are key mediators in the signaling pathways for apoptosis and cell disassembly (Thornberry, Chem. Biol., 1998, 5, R97-R103). There are twelve known human caspases, all of which cleave specifically at aspartyl residues and they all have stringent requirements for at least four amino acid residues on the N-terminal side of the cleavage site.
The caspases have been classified into three groups depending on the amino acid sequence that is preferred or primarily recognized. The first group of caspases, 1, 4, and 5, has been shown to prefer hydrophobic aromatic amino acids at position 4 on the N-terminal side of the cleavage site. A second group of caspases 2, 3 and 7, recognize aspartyl residues at both positions 1 and 4 on the N-terminal side of the cleavage site, and preferably a sequence of Asp-Glu-X-Asp. A third group of caspases 6, 8, 9 and 10, tolerate many amino acids in the primary recognition sequence.
A four amino acid sequence primarily recognized by the caspases has been determined for enzyme substrates (Talanian et al., J. Biol. Chem. 1997, 272, 9677-9682; Thornberry et al., J. Biol. Chem. 1997, 272, 17907-17911). Reversible tetrapeptide inhibitors have been prepared with the structure CH3CO—[P4]-[P3]-[P2]-CH(R)CH2CO2H where P2 to P4 represent an optimal amino acid recognition sequence and R is an aldehyde, nitrile or ketone capable of binding to the caspase cysteine sulfhydryl (Rano and Thornberry, Chem. Biol. 1997, 4, 149-155; Mjalli, et al., Bioorg. Med. Chem. Lett. 1993, 3, 2689-2692; Nicholson et al., Nature 1995, 376, 37-43). The utility of caspase inhibitors to treat a variety of mammalian disease states associated with an increase in cellular apoptosis has been demonstrated using peptidic caspase inhibitors. In general, the peptidic inhibitors described in the art are potent against certain caspase enzymes. Furthermore, the ability to design and employ substrates comprising radiolabeling agents that are also effective as caspase inhibitors to detect or treat these disease states is also desirable.
A number of medical diagnostic procedures, including Positron Emission Tomography (PET), and Single Photon Emission Computed Tomography (SPECT) utilize radiolabeled compounds. PET and SPECT are very sensitive techniques and require small quantities of radiolabeled compounds, called tracers. The labeled compounds are transported, accumulated and converted in vivo in exactly the same way as the corresponding non-radioactively labeled compounds. Tracers, or probes, can be radiolabeled with a radionuclide useful for PET imaging, such as 11C, 13N, 15O, 18F, 61Cu, 62Cu, 64Cu, 67Cu, 68Ga, 124I, 125I and 131I, or with a radionuclide useful for SPECT imaging, such as 99Tc, 75Br, 61Cu, 153Gd, 125I, 131I and 32P. One example of a PET probe is [18F]-fluorodeoxyglucose ([18F]-FDG).
PET creates images based on the distribution of molecular imaging tracers carrying positron-emitting isotopes in the tissue of the patient. The PET method has the potential to detect malfunction on a cellular level in the investigated tissues or organs. PET has been used in clinical oncology, such as for the imaging of tumors and metastases, and has been used for diagnosis of certain brain diseases, as well as mapping brain and heart function. Similarly, SPECT can be used to complement any gamma imaging study, where a true 3D representation can be helpful, for example, imaging tumor, infection (leukocyte), thyroid or bones.
The accurate detection of diseased tissue requires both spatial and biochemical feedback. For example, a two-step diagnosis involving both CT-based analysis and tissue biopsy guides clinicians in helping elucidate the presence and nature of a suspected disease. These two steps are necessary because CT analysis, devoid of any biochemical information, has limited benefit without complimentary information. In contrast, other imaging modalities can provide both spatial and biochemical information instantaneously. In vivo imaging of biochemical reporters provides critical biochemical information, deriving from the up- or down-regulation of specific cellular reporters, and in tandem, providing key spatial information. For instance, positron emission tomography (PET) imaging with 18F-FDG, routinely used by clinicians, accurately detects tumors and monitors tumor progression as a function of time.
18F-FDG imaging has broad clinical applications in detecting diseased tissue, such as tumors. However, 18F-FDG uptake in tumors strictly correlates with hexokinase activity, i.e., glucose metabolism, and thus 18F-FDG cannot provide critical information regarding a tumor's phenotype, receptor expression or potential to respond to a specific type of therapy. Thus, several tumor imaging approaches focus on employing small molecule ligands or small molecule substrates, for gathering a tumor's clinically relevant information. As an example, 18F-labeled estrogen analogs differentiate between ER+ and ER− breast tumors, which FDG cannot do, providing information regarding treatment plans involving the use of hormone-based therapies. Another tracer, 3′-Deoxy-3′-[18F]fluorothymidine (18[F]-FLT), effectively locates proliferating S-phase cells in brain gliomas, exceeding FDG in this application. In addition, 18F-fluoromisonidazole (18F-MISO) accurately targets hypoxic tumors which classically resist normal modes of cancer treatment, and helps guide specialized and effective therapeutic regimens. Clearly, a tumor's detection, characterization and its potential response to therapy provides critical information that guides therapeutic regimens that are more effective for the patient.
The vast majority of PET imaging agents are small molecule ligands that undergo facile radiolabeling, have optimized pharmacokinetic profiles, and efficiently localize to the target site. Unfortunately, they tend to function poorly in diseased tissue containing transiently expressed reporters or reporters expressed in low density, as the stoichiometric binding of ligand to target results in decreased signal output. Alternatively, small molecule substrate analogs useful for PET imaging, such as 18F-FDG and 18F-FLT, potentially offer enhanced signal amplification because of enzyme mediated intracellular turnover. Despite the gain in signal, the highly optimized and sensitive nature of the substrate-target interaction disallows major changes to the substrate scaffold making successful development of this class of agents notoriously difficult.
There are tracers that bind to reporters despite non-trivial modifications on the tracer. For example, radiolabeled peptide-based imaging agents possess high binding affinities and selectivities to their targets in vivo, yet these peptides bearing grossly modified chelating ligands appear to sustain their efficient binding affinities. While these tracers are not necessarily substrates for their targets, it is clear that despite their gross modifications, these agents function as effective tracers. Their success not withstanding, their usefulness as tracers is limited. Because of their size and overall electrostatic charge, they possess undesirable clearance half-lives, display poor metabolic profiles, and maintain poor cell permeability properties causing inefficient localization to intracellular reporters.
Consequently, it would be an advancement in the art to have improved imaging tracers which provide signal enhancement associated with substrate analogs in conjunction with specificity and generality associated with radiolabeled peptides. It would also be an advancement in the art to overcome difficulties in cellular transport and permeability while efficiently targeting intracellular reporters.